Three-dimensional reconstruction technology of the cryo-electron microscopy and X-ray crystallography, nuclear magnetic resonance spectrometry etc. have become the most important experiment methods for researching high resolution structural biology, more and more biological macromolecule structures are resolved successively. However, the eventual goal is to reveal the mechanism of the life activities on the different scales such as the molecule, organelle, cell by resolving the structures of biological macromolecule and molecular machine in situ.
In recent years, people may directly observe the positioning and motion of the target molecules in the cell and resolve the cell ultrastructure where the target molecules are by the use of the fluorescent protein labeling technology, the fluorescent microscopy, the immuno-electron microscopy, the cryo-electron microscopy and the electron tomography. With the development of the cell biology, simultaneously, the high precise positioning and the research of the high resolution ultrastructure for the molecular machines at the same position in the same cell using the fluorescent microscopy and the electron microscopy become the powerful research means, and this technology is referred to as Correlative Light and Electron Microscopy (CLEM). The targets are labeled and positioned by the fluorescent microscopy, the three-dimensional structure of the specified site of the cell is acquired by the three-dimensional reconstruction technology of the electron microscopes, and the positioning information and the structure information are merged and processed, thereby obtaining a number of the three-dimensional structure information regarding the molecular machines in the cells in situ to statistically achieve the dynamic variation mechanisms of the target molecular mechanisms in situ.
Currently, there are mainly two kinds of ways for the hardware implementations of correlative light and electron microscopy: one is an integrated system of the light microscope and electron microscope, that is, an optical imaging module is integrated into an electron microscope. The advantages lie in that it may be achieved that the light microscope and the electron microscope in situ of the biological specimen are imaged respectively and even imaged simultaneously, the image matching of the optical imaging and the electron imaging are convenient, and the cumbersome steps for transferring specimens between the light microscope and the electron microscope and the possibly caused contamination for the specimens are avoided. However, the small space between two pole pieces of objective lens of transmission electron microscope limited the optical imaging system put in it. Thus, the working distance of the objective lens must be larger, and it is difficult to obtain high resolution fluorescent images. Further, a more flexible correlative way is independent imaging of the light microscope and the electron microscope, a cryo-stage is mounted on independent optical imaging system, cryo-fluorescent imaging is accomplished, and then the specimen is transferred from the cryo-stage to the cryo-electron microscope for imaging. The advantages of this correlative way are in that the light microscope and electron microscope are unlimited with respect to each other in hardware, the modes of the optical imaging are various, which facilitates to achieve the higher accuracy fluorescent positioning. However, the design scheme of the existing cryo-stage mostly uses liquid nitrogen (or low temperature nitrogen) for flow refrigeration. The drifting of the specimen caused by the liquid nitrogen shaking and damaging of the objective lens in low temperature condition are all the difficult problem of affecting popularization of this technology. Meanwhile, the damage and contamination of the freezing specimen during the light microscope imaging and the transmission to the cryo-electron microscope is also a significant challenge.